Identification of formylglycine in sulfatases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

C(alpha)-Formylglycine, the catalytic amino acid residue in the active site of sulfatases, is generated by post-translational modification of a cysteine or serine residue. We describe a highly sensitive procedure for the detection of C(alpha)-formylglycine-containing peptides in tryptic digests of sulfatase proteins. The protocol is based on the formation of hydrazone derivatives of C(alpha)-formylglycine-containing peptides when using dinitrophenylhydrazine as a matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The hydrazone derivatives desorb and ionize with high efficiency and can be detected in the sub-femtomole range. The presence of C(alpha)-formylglycine is indicated by a mass increment of 180.13 u, corresponding to the hydrazone moiety, and also by a unique C-terminal fragment ion, characteristic of sulfatases, that becomes prominent in MALDI post-source decay mass spectra of the hydrazone derivatives.     

Purification,Characterization, and Structural Studies of a Sulfatase from Pedobacter yulinensis

Sulfatases are ubiquitous enzymes that hydrolyze sulfate from sulfated organic substrates such as carbohydrates, steroids, and flavones. These enzymes can be exploited in the field of biotechnology to analyze sulfated metabolites in humans, such as steroids and drugs of abuse. Because genomic data far outstrip biochemical characterization, the analysis of sulfatases from published sequences can lead to the discovery of new and unique activities advantageous for biotechnological applications. We expressed and characterized a putative sulfatase (PyuS) from the bacterium Pedobacter yulinensis. PyuS contains the (C/S)XPXR sulfatase motif, where the Cys or Ser is post-translationally converted into a formylglycine residue (FGly). His-tagged PyuS was co-expressed in Escherichia coli with a formylglycine-generating enzyme (FGE) from Mycobacterium tuberculosis and purified. We obtained several crystal structures of PyuS, and the FGly modification was detected at the active site. The enzyme has sulfatase activity on aromatic sulfated substrates as well as phosphatase activity on some aromatic phosphates; however, PyuS did not have detectable activity on 17α-estradiol sulfate, cortisol 21-sulfate, or boldenone sulfate.     

HPLC-MS/MS method to determine genipin in rat plasma after hydrolysis with sulfatase and its application to a pharmacokinetic study

A sensitive high‐performance liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed for the quantification of genipin in rat plasma after hydrolysis with sulfatase. Genipin could not be detected directly as it could be transformed into other forms such as conjugated‐genipin immediately after administration. The conjugated genipin could be hydrolyzed by sulfatase to genipin. The conditions of hydrolysis were investigated. Genipin and the internal standard, peoniflorin (IS), were separated on a reversed‐phase column by gradient elution and detected using an electrospray ion source on a 4000 QTrap triple‐quadrupole mass spectrometer. The quantification was performed using multiple reaction monitoring with selected precursor‐product ion pairs of the transitions m/z 225.0 → 122.7 and m/z 479.1 → 449.1 for genipin and peoniflorin. The assay was linear over the concentration range of 1.368–1368 ng/mL, with correlation coefficients of 0.9989. Intra‐ and inter‐day precisions and accuracy were all within 15%. The lower limit of quantification was 1.368 ng/mL. The recoveries of genipin and peoniflorin were more than 53.3 and 51.2%. The highly sensitive method was successfully applied to estimated pharmacokinetic parameters of genipin following oral and intravenous administration to rats. The absolute bioavailability of genipin was 80.2% in rat, which is the first report. Copyright © 2011 John Wiley & Sons, Ltd.     

Urinary Excretion of Disulfated Disaccharides in Hunter Syndrome: Correction by Infusion of a Serum Fraction

Abstract

The presence of disulfated disaccharides containing iduronic acid has been demonstrated in the urine of a Hunter patient by gel filtration of the low molecular weight urinary mucopolysaccharides. Intravenous infusion of a purified fraction from normal human serum causes disappearance of these metabolites which probably represent the substrate of the enzyme deficient in the disease, a sulfatase specific for an ester group situated on position 2 or 3 of the iduronic acid residues of dermatan sulfate.     

Simple Determination of Steroid Sulfatase Activity in Enzyme Preparation

Abstract

Simple one tube method for the determination of steroid sulfatase activity in an enzyme preparation or commercially available preparation is described. Principle of the reaction is as follows; dehydroepi-androsterone sulfate (DHEA-S) as substrate is hydrolyzed by the catalysis of sulfatase and DHEA released is determined by the enzymic method using 3β-hydroxysteroid oxidase.     

Stereoselective hydrolysis of sec-mono-alkyl sulfate esters with retention of configuration

An optimised method for the stereoselective hydrolysis of sec-alkylsulfate monoesters with absolute retention of configuration was developed. Under optimised conditions, clean hydrolysis of (R)-2-octyl sulfate was achieved in aqueous t-butyl methyl ether (3:97) using 0.6 equiv of p-toluenesulfonic acid as catalyst and 0.33 equiv of dioxane as mediator to give (R)-2-octanol as the sole product in the absence of side reactions, such as racemisation or elimination.